ABSTRACT Lenalidomide is a highly effective treatment for patients with myelodysplastic syndrome (MDS) with deletion of Chromosome 5q (del(5q)), with approximately 50% of patients achieving a complete cytogenetic response. In the previous funding period, we determined the mechanistic basis for the therapeutic efficacy of lenalidomide in del(5q) MDS, generated the first murine model that responds to lenalidomide, and developed a quantitative mass spectrometry-based approach to evaluate the activity of lenalidomide and related molecules. We now propose to investigate the molecular basis for incomplete responses to lenalidomide and acquired resistance, and to develop a novel therapeutic approach that bypasses resistance. First, we aim to determine mechanisms of sensitivity and resistance that have not been previously investigated: the impact of lenalidomide on stem cells, the bone marrow microenvironment, and the immune system. These experiments are enabled by the CRBNI391V murine model, developed during the previous funding period, that is sensitive to lenalidomide. Next, we will examine combinatorial targeting of CSNK1A1 and GSPT1 with a new thalidomide derivative, CC-885, and investigate the mechanism of GSPT1-mediated cytotoxicity in models of del(5q) MDS. Finally, we will investigate the biochemistry of lenalidomide-dependent degradation of oncoproteins by the CRL4CRBN E3 ubiquitin ligase, validating genes identified through genome-wide CRISPR screens. These studies will inform the biology of thalidomide derivatives in general, provide a comprehensive description and mechanistic understanding of thalidomide derivative effects on immune system homeostasis, and contribute to the development of novel therapeutics for del(5q) MDS.